Phytase Variants

ABSTRACT

The present invention relates to phytase variants, their preparation and uses, which phytase variants, when aligned according to FIG.  1 , are amended as compared to a model phytase in at least one of a number of positions. Preferred model phytases are basidiomycete and ascomycete phytases, such as  Peniophora  phytase and  Aspergillus  phytases. Preferred phytase variants exhibits amended activity characteristics, such as improved specific activity and/or improved thermostability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/734,510 filed Dec. 12, 2003, which is a divisional of U.S.application Ser. No. 10/083,452 filed Feb. 26, 2002, now U.S. Pat. No.6,689,358, which is a continuation of U.S. application Ser. No.09/273,871 filed Mar. 22, 1999, now U.S. Pat. No. 6,514,495, whichclaims priority or the benefit of Danish application nos. PA 1998 00407,PA 1998 00806, PA 1998 01176, and PA 1999 00091 filed Mar. 23, 1998,Jun. 19, 1998, Sep. 18, 1998 and Jan. 22, 1999, respectively, and U.S.provisional application Nos. 60/080,129, 60/090,675, 60/101,642 and60/117,677 filed Mar. 31, 1998, Jun. 25, 1998, Sep. 24, 1998 and Jan.28, 1999, respectively, the contents of which are fully incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to variants of phytases, in particular variantsof ascomycete phytases and variants of basidiomycete phytases, thecorresponding cloned DNA sequences, a method of producing such phytasevariants, and the use thereof for a number of industrial applications.

2. Description of Related Art

Phytic acid or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate (orfor short myo-inositol hexakisphosphate) is the primary source ofinositol and the primary storage form of phosphate in plant seeds.Phytin is a mixed potassium, magnesium and calcium salt of inositol.

The phosphate moieties of phytic acid chelates divalent and trivalentcations such as metal ions, i.a. the nutritionally essential ions ofcalcium, iron, zinc and magnesium as well as the trace mineralsmanganese, copper and molybdenum.

Phytic acid and its salts, phytates, are often not metabolized, i.e.,neither the phosphorous thereof, nor the chelated metal ions arenutritionally available.

Accordingly, food and feed preparations need to be supplemented withinorganic phosphate and often also the nutritionally essential ions suchas iron and calcium, must be supplemented.

Still further, the phytate phosphorus passes through thegastrointestinal tract of such animals and is excreted with the manure,resulting in an undesirable phosphate pollution of the environmentresulting e.g., in eutrophication of the water environment and extensivegrowth of algae.

Phytic acid or phytates, said terms being, unless otherwise indicated,in the present context used synonymously or at random, are degradable byphytases.

The production of phytases by plants as well as by microorganisms hasbeen reported. Amongst the microorganisms, phytase producing bacteria aswell as phytase producing fungi are known.

There are several descriptions of phytase producing filamentous fungibelonging to the fungal phylum of Ascomycota (ascomycetes). Inparticular, there are several references to phytase producingascomycetes of the Aspergillus genus such as Aspergillus terreus (Yamadaet al., 1986, Agric. Biol. Chem. 322:1275-1282). Also, the cloning andexpression of the phytase gene from Aspergillus niger var. awamori hasbeen described (Piddington et al., 1993, Gene 133:55-62). EP 0420358describes the cloning and expression of a phytase of Aspergillus ficuum(niger). EP 0684313 describes the cloning and expression of phytases ofthe ascomycetes Aspergillus niger, Myceliophthora thermophila,Aspergillus terreus. Still further, some partial sequences of phytasesof Aspergillus nidulans, Talaromyces thermophilus, Aspergillus fumigatusand another strain of Aspergillus terreus are given.

The cloning and expression of a phytase of Thermomyces lanuginosus isdescribed in WO 97/35017.

There is a current need for phytases of amended properties orcharacteristics, e.g., phytases of increased thermostability, altered pHoptimum (a high pH optimum being desirable for in-vitro processing, alow for in-vivo processing in the gastro-intestinal tract), and/or of ahigher specific activity.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides phytase variants, thecharacteristics of which are amended—as compared to a so-called modelphytase.

Any model phytase, which is of a certain similarity to thirteen hereinspecifically disclosed model phytases, can be made the model of suchvariants.

In another aspect, the invention relates to a novel phytase derived fromCladorrhinum foecundissimum.

In still another aspect, the invention provides DNA sequences encodingthese phytase variants and this phytase, and methods of theirproduction.

Finally, the invention also relates generally to the use of the phytaseand the phytase variants for liberating phosphorous from any phytasesubstrate, in particular inorganic phosphate from phytate or phyticacid.

BRIEF DESCRIPTION OF THE DRAWINGS

In the detailed description of the invention below, reference is made tothe drawings, of which

FIGS. 1A, 1B, 1C, and 1D show an alignment of thirteen specific phytasesequences (a multiple sequence alignment according to the programPileUp; GapWeight: 3.000; GapLengthWeight: 0.100) (SEQ ID NOS: 3-15);

FIG. 2 shows the amino acid and DNA sequence of a phytase (“C _(—)foecundissimum”) derived from strain CBS 427.97 of Cladorrhinumfoecundissimum (SEQ ID NOS: 1 and 2) which was deposited on 23 Jan.1997; the expression plasmid pYES 2.0 comprising the full length cDNAsequence was transformed into E. coli strain DSM 12742 which wasdeposited on 17 Mar. 1999;

FIG. 3 shows an alignment of the phytase C _(—) foecundissimum (SEQ IDNO: 2) with the model phytase M _(—) thermophila (SEQ ID NO: 15), usingthe program GAP gcg (Gap Weight 3.000; Length Weight 0.100); and

FIGS. 4A, 4B, 4C and 4D show how the C _(—) foecundissimum phytase canbe pasted onto the alignment of FIG. 1 (SEQ ID NOS: 2-15).

DETAILED DISCLOSURE OF THE INVENTION

Phytase

In the present context a phytase is an enzyme which catalyzes thehydrolysis of phytate (myo-inositol hexakisphosphate) to (1)myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphatesthereof and (3) inorganic phosphate. In the following, for short, theabove compounds are sometimes referred to as IP6, I, IP1, IP2, IP3, IP4,IP5 and P, respectively. This means that by action of a phytase, IP6 isdegraded into P+one or more of the components IP5, IP4, IP3, IP2, IP1and I. Alternatively, myo-inositol carrying in total n phosphate groupsattached to positions p, q, r, . . . is denoted Ins(p,q,r, . . . )Pn.For convenience Ins(1,2,3,4,5,6)P6 (phytic acid) is abbreviated PA.

According to the Enzyme nomenclature database ExPASy (a repository ofinformation relative to the nomenclature of enzymes primarily based onthe recommendations of the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (IUBMB) describing each typeof characterized enzyme for which an EC (Enzyme Commission) number hasbeen provided), two different types of phytases are known: A so-called3-phytase (myo-inositol hexaphosphate 3-phosphohydrolase, EC 3.1.3.8)and a so-called 6-phytase (myo-inositol hexaphosphate6-phosphohydrolase, EC 3.1.3.26). The 3-phytase hydrolyzes first theester bond at the D-3-position, whereas the 6-phytase hydrolyzes firstthe ester bond at the D-6- or L-6-position.

The expression “phytase” or “polypeptide or enzyme exhibiting phytaseactivity” is intended to cover any enzyme capable of effecting theliberation of inorganic phosphate or phosphorous from variousmyo-inositol phosphates. Examples of such myo-inositol phosphates(phytase substrates) are phytic acid and any salt thereof, e.g., sodiumphytate or potassium phytate or mixed salts. Also any stereoisomer ofthe mono-, di-, tri-, tetra- or penta-phosphates of myo-inositol mightserve as a phytase substrate. A preferred phytase substrate is phyticacid and salts thereof.

In accordance with the above definition, the phytase activity can bedetermined using any assay in which one of these substrates is used. Inthe present context (unless otherwise specified) the phytase activity isdetermined in the unit of FYT, one FYT being the amount of enzyme thatliberates 1 micro-mol inorganic ortho-phosphate per min. under thefollowing conditions: pH 5.5; temperature 37° C.; substrate: sodiumphytate (C₆H₆O₂₄P₆Na₁₂) in a concentration of 0.0050 mol/l. A suitablephytase assay is described in the experimental part.

The present invention provides a genetically engineered phytase asdescribed in the appending claims.

A genetically engineered phytase is a non-naturally occurring phytasewhich is different from a model phytase, e.g., a wild-type phytase.Genetically engineered phytases include, but are not limited to,phytases prepared by site-directed mutagenesis, gene shuffling, randommutagenesis etc.

The invention also provides DNA constructs, vectors, host cells, andmethods of producing these genetically engineered phytases and phytasevariants, as well as uses thereof.

A phytase variant is a polypeptide or enzyme or a fragment thereof whichexhibits phytase activity and which is amended as compared to a modelphytase.

Amended means altered by way of one or more amino acid or peptidesubstitutions, deletions, insertions and/or additions—in each case by,or of, one or more amino acids. Such substitutions, deletions,insertions, additions can be achieved by any method known in the art,e.g., gene shuffling, random mutagenesis, site-directed mutagenesis etc.

The model or parent phytase, from which the phytase variant is derived,can be any phytase, e.g., a wild-type phytase or a derivative, mutant orvariant thereof, including allelic and species variants, as well asgenetically engineered variants thereof, which e.g., can be prepared bysite-directed mutagenesis, random mutagenesis, shuffling etc.

Included in the concept of model phytase is also any hybrid or chimericphytase, i.e., a phytase which comprises a combination of partial aminoacid sequences derived from at least two phytases.

The hybrid phytase may comprise a combination of partial amino acidsequences deriving from at least two ascomycete phytases, at least twobasidiomycete phytases or from at least one ascomycete and at least onebasidiomycete phytase. These ascomycete and basidiomycete phytases fromwhich a partial amino acid sequence derives may, e.g., be any of thosespecific phytases referred to herein.

In the present context, a hybrid, shuffled, random mutagenized,site-directed mutagenized or otherwise genetically engineered phytasederived from ascomycete phytases only is also an ascomycete phytase; anda hybrid, shuffled, random mutagenized, site-directed mutagenized orotherwise genetically engineered phytase derived from modelbasidiomycete phytases only is also a basidiomycete phytase. Any hybridderived from at least one ascomycete phytase as well as at least onebasidiomycete phytase is called a mixed ascomycete/basidiomycete phytaseand such phytase is also a model phytase in the present context.

Analogously, a hybrid, shuffled, random mutagenized, site-directedmutagenized or otherwise genetically engineered phytase derived from oneor more Aspergillus phytases is also an Aspergillus derived phytase; anda hybrid, shuffled, random mutagenized, site-directed mutagenized orotherwise genetically engineered phytase derived from any othertaxonomic sub-grouping mentioned herein is also to be designated aphytase derived from this taxonomic sub-grouping.

Still further, in the present context, “derived from” is intended toindicate a phytase produced or producible by a strain of the organism inquestion, but also a phytase encoded by a DNA sequence isolated fromsuch strain and produced in a host organism transformed with said DNAsequence. Finally, the term is intended to indicate a phytase which isencoded by a DNA sequence of synthetic and/or cDNA origin and which hasthe identifying characteristics of the phytase in question.

Preferably the model phytase is a phytase which can be aligned asdescribed below to either of the thirteen phytases of FIG. 1 (which areparticularly preferred model phytases).

Preferred wild-type model phytases (i.e., neither recombinant, orshuffled or otherwise genetically engineered phytases) have a degree ofsimilarity or homology, preferably identity, to amino acid residues38-403 (Peniophora numbers) of either of these thirteen phytases or atleast 40%, more preferably at least 50%, still more preferably at least60%, in particular at least 70%, especially at least 80%, and in a mostpreferred embodiment a degree of similarity of at least 90%.

Preferred recombinant or shuffled or otherwise genetically engineeredmodel phytases have a degree of similarity or homology, preferablyidentity, to amino acid residues 38-49, 63-77, 274-291, 281-300 and389-403 (Peniophora numbers) of either of these thirteen phytases or atleast 60%, more preferably at least 70%, still more preferably at least80%, in particular at least 90%.

In a preferred embodiment the degree of similarity is based on acomparison with the complete amino acid sequence of either of thethirteen phytases.

The degree of similarity or homology, alternatively identity, can bedetermined using any alignment programme known in the art. A preferredalignment programme is GAP provided in the GCG version 8 program package(Program Manual for the Wisconsin Package, Version 8, August 1994,Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711)(see also Needleman, S. B. and Wunsch, C. D., (1970), Journal ofMolecular Biology, 48, 443-453). Using GAP with the following settingsfor polypeptide sequence comparison: GAP weight of 3.000 and GAP lengthweight of 0.100.

Also preferred is a wild-type model phytase which comprises an aminoacid sequence encoded by a DNA sequence which hybridizes to a DNAsequence encoding amino acid sequence 38-403 (Peniophora numbers) of anyof the DNA sequences encoding the thirteen specific phytase sequences ofFIG. 1.

A further preferred model phytase is a genetically engineered phytase,which comprises an amino acid sequence encoded by a DNA sequence whichhybridizes to a DNA sequence encoding amino acid sequence 38-49, and toa DNA sequence encoding amino acid sequence 63-77, and to a DNA sequenceencoding amino acid sequence 274-291, and to a DNA sequence encodingamino acid sequence 281-300, and to a DNA sequence encoding amino acidsequence 389-403 (Peniophora numbers) of any of the DNA sequencesencoding the thirteen specific phytase sequences of FIG. 1.

In a preferred embodiment the hybridization is to the complete phytaseencoding part of any of the thirteen phytases.

Suitable experimental conditions for determining whether a given DNA orRNA sequence “hybridizes” to a specified nucleotide or oligonucleotideprobe involves presoaking of the filter containing the DNA fragments orRNA to examine for hybridization in 5×SSC (Sodium chloride/Sodiumcitrate), (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.) for10 min, and prehybridization of the filter in a solution of 5×SSC,5×Denhardt's solution (Sambrook et al., 1989), 0.5% SDS and 100micrograms/ml of denatured sonicated salmon sperm DNA (Sambrook et al.,1989), followed by hybridization in the same solution containing aconcentration of 10 ng/ml of a random-primed (Feinberg, A. P. andVogelstein, B., 1983, Anal. Biochem. 132:6-13), ³²P-dCTP-labeled(specific activity>1×109 cpm/microgram) probe for 12 hours atapproximately 45° C.

The filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDS atleast 55° C. (low stringency), at least 60° C. (medium stringency), atleast 65° C. (medium/high stringency), at least 70° C. (highstringency), or at least 75° C. (very high stringency).

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using an x-ray film.

It should be noted that a certain specific phytase variant need notactually have been prepared from a specific model phytase, for thismodel phytase to qualify as a “model phytase” in the present context. Itis sufficient that the variant exhibits at least one of the hereinindicated amendments when it is afterwards compared with the modelphytase.

The alignment of FIG. 1 is made using the program PileUp (Program Manualfor the Wisconsin Package, Version 8, August 1994, Genetics ComputerGroup, 575 Science Drive, Madison, Wis., USA 53711), with a GapWeight of3.000 and a GapLengthWeight of 0.100. When aligning a new model phytaseor a new phytase variant all thirteen sequences can be included togetherwith the new phytase (variant) in a multiple alignment, or,alternatively, at least one of the thirteen sequences of FIG. 1 isincluded together with the new phytase (variant) in an alignment.

A preferred procedure for aligning according to FIG. 1 a new modelphytase (or a phytase variant) is as follows: The new model phytase isaligned with that specific sequence of the thirteen sequences of FIG. 1to which the new model phytase has the highest degree of homology. Forcalculating the degree of homology, and for making the “alignmentaccording to FIG. 1” of the two sequences, the program GAP referred tobelow is preferably used. Having aligned the two sequences, the newmodel phytase (or phytase variant) is added (pasted) to the alignment atFIG. 1 using the result of the first alignment (placing identical andhomologous amino acid residues above each other as prescribed by thealignment), following which corresponding positions are now easilyidentifiable.

Example 7 shows an example of how to add a new model phytase to thealignment of FIG. 1 and deduce corresponding phytase variants thereof.

Other model phytases can be aligned and variants deduced in analogy withExample 7. This is so in particular for the following model phytases:The phytase of Aspergillus niger var. awamori (U.S. Pat. No. 5,830,733);the Bacillus phytase of WO 98/06858; the soy bean phytase of WO98/20139; the maize phytase of WO 98/05785; the Aspergillus phytase ofWO 97/38096; the phytases of Monascus anka of WO 98/13480; the phytasefrom Schwanniomyces occidentalis of EP 0699762 etc.

When comparing a model phytase and a proposed phytase variant using thealignment as described herein, corresponding amino acid positions can beidentified, viz. a model position of the model phytase and a variantposition of the variant—the corresponding model position and variantposition are simply placed one above the other in the alignment. Anamendment is said to have occurred in a given position if the modelamino acid of the model position and the variant amino acid of thevariant position are different. Preferred amendments of these positionsmanifest themselves as amino acid substitutions, deletions or additions.

Amended in at least one position means amended in one or more positions,i.e., in one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve etc. up to all N positions listed. This definitionincludes any possible sub-combinations thereof, e.g., any set of twosubstitutions, any set of three, any set of four, etc.—to any set of(N−1) positions.

In the present context all sequences, whatever the model phytase, andincluding the thirteen sequences of FIG. 1, are numbered using thenumbering corresponding to the phytase P _(—) lycii. These “Peniophoranumbers” are indicated at FIG. 1, together with the “alignment numbers.”The numbering of P _(—) lycii starts at M1 and ends at E439.

As explained above, the alignment reveals which positions in variousphytase sequences other than P _(—) lycii are equivalent orcorresponding to the given P. lycii position.

A substitution of amino acids is indicated herein as for instance “3S,”which indicates, that at position 3 amino acid S should be substitutedfor the “original” or model position 3 amino acid, whichever it is.Thus, the substitution should result in an S in the correspondingvariant position. Considering now the alignment at FIG. 1, asubstitution like e.g., “3S” is to be interpreted as follows, for therespective phytases shown (the amino acid first indicated is the“original” or model amino acid in “Peniophora position” 3):

P_involtus_A1: F3S (number 3 F substituted by S) P_involtus_A2: L3ST_pubescens: M1S A_pediades: M1S P_lycii: redundant (already an S)A_fumigatus: T5S consphyA: V5S A_nidulans: T5S A_ficuum_NRRL3135: A5SA_terreus: A5S T_thermo: L5S T_lanuginosa: V11S M_thermophila: G5S

However, in what follows the above specific substitutions will bedesignated as follows (always using the Peniophora numbering):

P_involtus_A1: F3S P_involtus_A2: L3S T_pubescens: M3S A_pediades: M3SP_lycii: redundant (already an S) A_fumigatus: T3S consphyA: V3SA_nidulans: T3S A_ficuum_NRRL3135: A3S A_terreus: A3S T_thermo: L3ST_lanuginosa: V3S M_thermophila: G3S

Still further, denotations like e.g., “3S,F,G” means that the amino acidin position 3 (Peniophora numbers) of the model phytase in question issubstituted with either of S, F or G, i.e., e.g., the designation“3S,F,G” is considered fully equivalent to the designation “3S, 3F, 3G”.

A denotation like ( )3S means that amino acid S is added to the sequencein question (at a gap in the actual sequence), in a positioncorresponding to Peniophora number 3—and vice versa for deletions (S3()).

In case of regions in which the Peniophora phytase sequence has largerdeletions than some of the other phytases in FIG. 1, for instance in theregion between position 201 and 202 (Peniophora numbers), intermediatepositions (amino acid residues in other sequences) are numbered byadding a,b,c,d, etc, in lower-case letters, to the last Peniophoraposition number, e.g., for the phytase M _(—) thermophila: E201; G201a;P201b; Y201c; S201d; T201e; I201f; G202; D203 etc.

In one of the priority applications of the present application there aretwo minor position numbering errors: According to the above definitions,the positions referred to in the first priority application as 204 and205 (Peniophora numbers) are wrongly designated; they should have beennumbered 203a and 204, respectively. Therefore, 204 has been substitutedby 203a and 205 by 204 throughout the present application.

A preferred phytase variant of the invention comprises an amino acidsequence which comprises, preferably contains, one or more of thefollowing amino acid substitutions: 24C; 27P; 31Y; 33C; 39H,S,Q; 40L,N;42S,G; 43A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 44N; 45D,S; 47Y,F;49P; 51E,A,R; 56P; 58D,K,A; 59G; 61R; 62V,I; 69Q; 75W,F; 78D,S; 79G;80K,A; 81A,G,Q,E; 82T; 83A,I,K,R,Q; 84I,Y,Q,V; 88I; 90R,A; 102Y; 115N;116S; 118V,L; 119E; 120L; 122A; 123N,Q,T; 125M,S; 126H,S,V; 127Q,E,N;128A,S,T; 132F,I,L; 143N; 148V,I; 151A,S; 152G; 153D,Y; 154D,Q,S,G;157V; 158D,A; 159T; 160A,S; 161T,N; 162N; 163W; 170fH; 170gA; 171N;172P; 173Q,S; 184Q,S,P; 185S; 186A,E,P; 187A; 187aS; 190A,P; 193S;194S,T; 195T,V,L; 198A,N,V; 200G,V; 201D,E; a deletion of at least oneof 201a, 201b, 201c, 201d, 201e, 201f, preferably all; 201eT; 202S,A;203R,K,S; 203aV,T; 204Q,E,S,A,V; 205E; 211L,V; 215A,P; 220L,N; 223H,D;228N; 232T; 233E; 235Y,L,T; 236Y,N; 237F; 238L,M; 242P,S; 244D; 246V;251eE,Q; 253P; 256D; 260A,H; 264R,I; 265A,Q; 267D; 270Y,A,L,G; 271D,N;273D,K; 275F,Y; 278T,H; 280A,P; 283P; 287A,T; 288L,I,F; 292F,Y; 293A,V;302R,H; 304P,A; 332F; 336S; 337T,G,Q,S; 338I; 339V,I; 340P,A;343A,S,F,I,L; 348Y; 349P; 352K; 360R; 362P; 364W,F; 365V,L,A,S;366D,S,V; 367A,K; 368K; 369I,L; 370V; 373A,S; 374S,A; 375H; 376M;383kQ,E; 387P; 393V; 396R; 404A,G; 409R; 411K,T; 412R; 417E,R; 421F,Y;431E.

In a preferred embodiment this is with the proviso that the modelphytase does not already comprise the above suggested amino acidsubstitution or addition or deletion at the position indicated. Or, withthe proviso that, for each position, the model amino acid is not alreadythe variant amino acid hereby proposed. But these provisos can be saidto be in fact already inherent in the above wording, because of theexpression “amended.”

The various preferred phytase variants of claims 16-34 comprises,preferably contains or have, amino acid sequences which comprise orcontain one or more of the amino acid substitutions, additions, ordeletions listed in the respective claims.

In a preferred embodiment the various phytase variants comprise 1, 2, 3,4, 5, 6, 7, 8, 9 or even 10 of these substitutions; or a number ofsubstitutions of 10-15, 15-20, 20-30 or even 30-50; eventually up to 60,70, 80 or 90 substitutions.

In another preferred embodiment, the amino acid sequence of the variousphytase variants comprise one or more substitutions of the substitutionsub-groupings listed hereinbelow; or combinations of substitutionsclassified in two or more sub-groupings.

Generally, instead of “comprise,” “contain” or “have,” the amino acidsequences of preferred variants “consist essentially of” or “consist of”the specific model phytases of FIG. 1, as modified by one or more of thesubstitutions described herein.

In the present context a basidiomycete means a microorganism of thephylum Basidiomycota. This phylum of Basidiomycota is comprised in thefungal kingdom together with e.g., the phylum Ascomycota(“ascomycetes”).

Taxonomical questions can be clarified by consulting the referenceslisted below or by consulting a fungal taxonomy database (NIH Data Base(Entrez)) which is available via the Internet on World Wide Web.

For a definition of basidiomycetes, reference is made to either Jülich,1981, Higher Taxa of Basidiomycetes; Ainsworth & Bisby's (eds.)Dictionary of the Fungi, 1995, Hawksworth, D. L., P. M. Kirk, B. C.Sutton & D. N. Pegler; or Hansen & Knudsen (Eds.), Nordic Macromycetes,vol. 2 (1992) and 3 (1997). A preferred reference is Hansen & Knudsen.

For a definition of ascomycetes, reference is made to either ofAinsworth & Brisby cited above or Systema Ascomycetum by Eriksson, O. E.& D. L. Hawksworth, Vol. 16, 1998. A preferred reference is Eriksson etal.

Generally, a microorganism which is classified as abasidiomycete/ascomycete in either of the references listed above,including the database, is a basidiomycete/ascomycete in the presentcontext.

Some Aspergillus strains are difficult to classify because they areanamorphous, and therefore they might be classified in Fungi Imperfecti.However, once the teleomorphous counterpart is found, it isre-classified taxonomically. For instance, the teleomorph of A. nidulansis Emericella nidulans (of the family Trichocomaceae, the orderEurotiales, the class Plectomycetes of the phylum Ascomycota). Thesesubgroupings of Ascomycota are preferred, together with the familyLasiosphaeriaceae, the order Sordariales, the class Pyrenomycetes of thephylum Ascomycota.

The wording “ascomycetes” and analogues as used herein includes anystrains of Aspergillus, Thermomyces, Myceliophthora, and Talaromyces,which are anamorphous and thus would be classified in Fungi Imperfecti.

Preferred basidiomycete phytases are those listed in WO 98/28409, in thevery beginning of the section headed “Detailed description of theinvention”.

DNA sequences encoding the thirteen specifically listed model phytasesand other model phytases can be prepared according to the teachings ofeach of the documents listed under the brief description of thedrawings.

A DNA sequence encoding a model phytase may be isolated from any cell ormicroorganism producing the phytase in question, using various methodswell known in the art. First, a genomic DNA and/or cDNA library shouldbe constructed using chromosomal DNA or messenger RNA from the organismthat produces the phytase. Then, if the amino acid sequence of thephytase is known, homologous, labelled oligonucleotide probes may besynthesized and used to identify phytase-encoding clones from a genomiclibrary prepared from the organism in question. Alternatively, alabelled oligonucleotide probe containing sequences homologous to aknown phytase gene could be used as a probe to identify phytase-encodingclones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying phytase-encoding clones would involveinserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming phytase-negative bacteria with the resultinggenomic DNA library, and then plating the transformed bacteria onto agarcontaining a substrate for phytase thereby allowing clones expressingthe phytase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers(1981) or the method described by Matthes et al. (1984). In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or R. K.Saiki et al. (1988).

DNA encoding the phytase variants of the present invention can beprepared by methods known in the art, such as site-directed mutagenesis.Once a DNA sequence encoding a model phytase of interest has beenisolated, and desirable sites for mutation identified, mutations may beintroduced using synthetic oligonucleotides. These oligonucleotidescontain nucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging the phytase-encodingsequence, is created in a vector carrying the phytase-encoding gene.Then the synthetic nucleotide, bearing the desired mutation, is annealedto a homologous portion of the single-stranded DNA. The remaining gap isthen filled in with DNA polymerase I (Klenow fragment) and the constructis ligated using T4 ligase. A specific example of this method isdescribed in Morinaga et al. (1984). U.S. Pat. No. 4,760,025 disclosesthe introduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette. However, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod because a multitude of oligonucleotides, of various lengths, canbe introduced.

Another method of introducing mutations into DNA sequences encoding adesired model phytase is described in Nelson and Long (1989). Itinvolves a 3-step generation of a PCR fragment containing the desiredmutation introduced by using a chemically synthesized DNA strand as oneof the primers in the PCR reactions. From the PCR-generated fragment, aDNA fragment carrying the mutation may be isolated by cleavage withrestriction endonucleases and reinserted into an expression plasmid.

Yet another method of mutating DNA sequences encoding a model phytase israndom mutagenesis. Random mutagenesis is suitably performed either aslocalized or region-specific random mutagenesis in at least three partsof the gene translating to the amino acid sequence shown in question, orwithin the whole gene.

The random mutagenesis of a DNA sequence encoding a model phytase may beconveniently performed by use of any method known in the art.

In relation to the above, further aspects of the present inventionrelates to a method for generating a variant of a model phytase, whereinthe variant preferably exhibits amended characteristics as describedbelow, the method comprising:

(a) subjecting a DNA sequence encoding the model phytase toSite-directed Mutagenesis, or the Nelson and Long PCR mutagenesis methodor to random mutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a hostcell, and

(c) screening for host cells expressing a phytase variant which has analtered property relative to the model phytase.

When using random mutagenesis, step (a) of the above method of theinvention is preferably performed using doped primers.

For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues. When such agents are used, themutagenesis is typically performed by incubating the DNA sequenceencoding the parent enzyme to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

When the mutagenesis is performed by the use of an oligonucleotide, theoligonucleotide may be doped or spiked with the three non-parentnucleotides during the synthesis of the oligonucleotide at the positionswhich are to be changed. The doping or spiking may be done so thatcodons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding the phytaseenzyme by any published technique, using e.g., PCR, LCR or any DNApolymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints. The doping scheme may be made byusing the DOPE program which, inter alia, ensures that introduction ofstop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated ornon-treated gene encoding a model phytase is subjected to PCR underconditions that increase the mis-incorporation of nucleotides (Deshler,1992; Leung et al., 1989, Technique, Vol. 1, pp. 11-15).

A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133,1974, pp. 179-191), S. cereviseae or any other microbial organism may beused for the random mutagenesis of the DNA encoding the model phytaseby, e.g., transforming a plasmid containing the parent glycosylase intothe mutator strain, growing the mutator strain with the plasmid andisolating the mutated plasmid from the mutator strain. The mutatedplasmid may be subsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in agenomic or cDNA library prepared from an organism expressing the modelphytase. Alternatively, the DNA sequence may be present on a suitablevector such as a plasmid or a bacteriophage, which as such may beincubated with or otherwise exposed to the mutagenizing agent. The DNAto be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharboured in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

In some cases it may be convenient to amplify the mutated DNA sequenceprior to performing the expression step b) or the screening step c).Such amplification may be performed in accordance with methods known inthe art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenizing agent,the mutated DNA is expressed by culturing a suitable host cell carryingthe DNA sequence under conditions allowing expression to take place. Thehost cell used for this purpose may be one which has been transformedwith the mutated DNA sequence, optionally present on a vector, or onewhich was carried the DNA sequence encoding the parent enzyme during themutagenesis treatment. Examples of suitable host cells are thefollowing: gram positive bacteria such as Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearotherinophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encodingfunctions permitting expression of the mutated DNA sequence.

The random mutagenesis may be advantageously localized to a part of themodel phytase in question using Localized random mutagenesis. This may,e.g., be advantageous when certain regions of the enzyme have beenidentified to be of particular importance for a given property of theenzyme, and when modified are expected to result in a variant havingimproved properties. Such regions may normally be identified when thetertiary structure of the parent enzyme has been elucidated and relatedto the function of the enzyme.

The localized or region-specific random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

For region-specific random mutagenesis with a view to amending e.g., thespecific activity of a model phytase, codon positions corresponding tothe following amino acid residues from the amino acid sequences setforth in FIG. 1 may appropriately be targeted:

Residues: 41-47, 68-80, 83-84, 115-118, 120-126, 128, 149-163, 184-185,191-193, 198-201e, 202-203, 205, 235-236, 238-239, 242-243, 270-279,285, 288, 332-343, 364-367, 369-375, 394.

Regions: 41-47, 68-80, 120-128, 149-163, 270-279, 332-343, 364-375.

The random mutagenesis may be carried out by the following steps:

1. Select regions of interest for modification in the parent enzyme

2. Decide on mutation sites and non-mutated sites in the selected region

3. Decide on which kind of mutations should be carried out, e.g., withrespect to the desired stability and/or performance of the variant to beconstructed

4. Select structurally reasonable mutations

5. Adjust the residues selected by step 3 with regard to step 4.

6. Analyse by use of a suitable dope algorithm the nucleotidedistribution.

7. If necessary, adjust the wanted residues to genetic code realism,e.g., taking into account constraints resulting from the genetic code,e.g., in order to avoid introduction of stop codons; the skilled personwill be aware that some codon combinations cannot be used in practiceand will need to be adapted

8. Make primers

9. Perform random mutagenesis by use of the primers

10. Select resulting phytase variants by screening for the desiredimproved properties.

Suitable dope algorithms for use in step 6 are well known in the art.One such algorithm is described by Tomandl, D. et al., 1997, Journal ofComputer-Aided Molecular Design 11:29-38. Another algorithm is DOPE(Jensen, L J, Andersen, K V, Svendsen, A, and Kretzschmar, T., 1998,Nucleic Acids Research 26:697-702).

A DNA sequence encoding a model phytase or a phytase variant of theinvention can be expressed using an expression vector, a recombinantexpression vector, which typically includes control sequences encoding apromoter, operator, ribosome binding site, translation initiationsignal, and, optionally, a repressor gene or various activator genes.

The recombinant expression vector may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,e.g., a plasmid, a bacteriophage or an extra-chromosomal element.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. An example of a suitable promoter for directing thetranscription of the DNA sequence encoding a phytase variant of theinvention, especially in a bacterial host, is the promoter of the lacoperon of E. coli. For transcription in a fungal host, examples ofuseful promoters are those derived from the gene encoding A. oryzae TAKAamylase.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the phytase variant ofthe invention. Termination and polyadenylation sequences may suitably bederived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin resistance. Furthermore, thevector may comprise Aspergillus selection markers such as amdS, argB,niaD and sC, or the selection may be accomplished by co-transformation,e.g., as described in WO 91/17243.

The procedures used to ligate the DNA construct of the inventionencoding a phytase variant, the promoter, terminator and other elements,respectively, and to insert them into suitable vectors containing theinformation necessary for replication, are well known to persons skilledin the art (cf., for instance, Sambrook et al. (1989)).

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of a phytase variantof the invention. The cell may be transformed with the DNA construct ofthe invention encoding the variant, conveniently by integrating the DNAconstruct (in one or more copies) in the host chromosome. Thisintegration is generally considered to be an advantage as the DNAsequence is more likely to be stably maintained in the cell. Integrationof the DNA constructs into the host chromosome may be performedaccording to conventional methods, e.g., by homologous or heterologousrecombination. Alternatively, the cell may be transformed with anexpression vector as described above in connection with the differenttypes of host cells.

An isolated DNA molecule or, alternatively, a “cloned DNA sequence” “aDNA construct,” “a DNA segment” or “an isolated DNA sequence” refers toa DNA molecule or sequence which can be cloned in accordance withstandard cloning procedures used in genetic engineering to relocate theDNA segment from its natural location to a different site where it willbe replicated. The term refers generally to a nucleic acid sequencewhich is essentially free of other nucleic acid sequences, e.g., atleast about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by agarose gel electrophoresis. The cloning procedures mayinvolve excision and isolation of a desired nucleic acid fragmentcomprising the nucleic acid sequence encoding the polypeptide, insertionof the fragment into a vector molecule, and incorporation of therecombinant vector into a host cell where multiple copies or clones ofthe nucleic acid sequence will be replicated. The nucleic acid sequencemay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The term “vector” is intended to include such terms/objects as “nucleicacid constructs,” “DNA constructs,” expression vectors” or “recombinantvectors.”

The nucleic acid construct comprises a nucleic acid sequence of thepresent invention operably linked to one or more control sequencescapable of directing the expression of the coding sequence in a suitablehost cell under conditions compatible with the control sequences.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature.

The term nucleic acid construct may be synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of thepresent invention.

The term “coding sequence” as defined herein primarily comprises asequence which is transcribed into mRNA and translated into apolypeptide of the present invention when placed under the control ofthe above mentioned control sequences. The boundaries of the codingsequence are generally determined by a translation start codon ATG atthe 5′-terminus and a translation stop codon at the 3′-terminus. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for expression of the codingsequence of the nucleic acid sequence. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader, apolyadenylation sequence, a propeptide sequence, a promoter, a signalsequence, and a transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide.

A “host cell” or “recombinant host cell” encompasses any progeny of aparent cell which is not identical to the parent cell due to mutationsthat occur during replication.

The cell is preferably transformed with a vector comprising a nucleicacid sequence of the invention followed by integration of the vectorinto the host chromosome.

“Transformation” means introducing a vector comprising a nucleic acidsequence of the present invention into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector. Integration is generally considered to be anadvantage as the nucleic acid sequence is more likely to be stablymaintained in the cell. Integration of the vector into the hostchromosome may occur by homologous or non-homologous recombination asdescribed above.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote. Examples ofeukaryote cells are a mammalian cell, an insect cell, a plant cell or afungal cell. Useful mammalian cells include Chinese hamster ovary (CHO)cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or anynumber of other immortalized cell lines available, e.g., from theAmerican Type Culture Collection.

In a preferred embodiment, the host cell is a fungal cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se.

The present invention also relates to a transgenic plant, plant part,such as a plant seed, or plant cell, which has been transformed with aDNA sequence encoding the phytase of the invention so as to express orproduce this enzyme. Also compositions and uses of such plant or plantpart are within the scope of the invention, especially its use as feedand food or additives therefore, along the lines of the present use andfood/feed claims.

The transgenic plant can be dicotyledonous or monocotyledonous, forshort a dicot or a monocot. Of primary interest are such plants whichare potential food or feed components and which comprise phytic acid. Anormal phytic acid level of feed components is 0.1-100 g/kg, or moreusually 0.5-50 g/kg, most usually 0.5-20 g/kg. Examples of monocotplants are grasses, such as meadow grass (blue grass, Poa), forage grasssuch as festuca, lolium, temperate grass, such as Agrostis, and cereals,e.g., wheat, oats, rye, barley, rice, sorghum and maize (corn).

Examples of dicot plants are legumes, such as lupins, pea, bean andsoybean, and cruciferous (family Brassicaceae), such as cauliflower, oilseed rape and the closely related model organism Arabidopsis thaliana.

Such transgenic plant etc. is capable of degrading its own phytic acid,and accordingly the need for adding such enzymes to food or feedcomprising such plants is alleviated. Preferably, the plant or plantpart, e.g., the seeds, are ground or milled, and possibly also soakedbefore being added to the food or feed or before the use, e.g., intake,thereof, with a view to adapting the speed of the enzymatic degradationto the actual use.

If desired, the plant produced enzyme can also be recovered from theplant. In certain cases the recovery from the plant is to be preferredwith a view to securing a heat stable formulation in a potentialsubsequent pelleting process.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,tubers etc. But also any plant tissue is included in this definition.

Any plant cell, whatever the tissue origin, is included in thedefinition of plant cells above.

Also included within the scope of the invention are the progeny of suchplants, plant parts and plant cells.

The skilled man will know how to construct a DNA expression constructfor insertion into the plant in question, paying regard i.a. to whetherthe enzyme should be excreted in a tissue specific way. Of relevance forthis evaluation is the stability (pH-stability, degradability byendogenous proteases etc.) of the phytase in the expression compartmentsof the plant. He will also be able to select appropriate regulatorysequences such as promoter and terminator sequences, and signal ortransit sequences if required (Tague et al, Plant, Phys., 86, 506,1988).

The plant, plant part etc. can be transformed with this DNA constructusing any known method. An example of such method is the transformationby a viral or bacterial vector such as bacterial species of the genusAgrobacterium genetically engineered to comprise the gene encoding thephytase of the invention. Also methods of directly introducing thephytase DNA into the plant cell or plant tissue are known in the art,e.g., micro injection and electroporation (Gasser et al., Science, 244,1293; Potrykus, Bio/Techn. 8, 535, 1990; Shimamoto et al., Nature, 338,274, 1989).

Following the transformation, the transformants are screened using anymethod known to the skilled man, following which they are regeneratedinto whole plants.

These plants etc. as well as their progeny then carry the phytaseencoding DNA as a part of their genetic equipment.

In general, reference is made to WO 91/14782 and WO 91/14772.

Agrobacterium tumefaciens mediated gene transfer is the method of choicefor generating transgenic dicots (for review Hooykas & Schilperoort,1992, Plant Mol. Biol. 19: 15-38), however it can also be used fortransforming monocots. Due to host range limitations it is generally notpossible to transform monocots with the help of A. tumefaciens. Here,other methods have to be employed. The method of choice for generatingtransgenic monocots is particle bombardment (microscopic gold ortungsten particles coated with the transforming DNA) of embryonic callior developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto,1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674).

Also other systems for the delivery of free DNA into these plants,including viral vectors (Joshi & Joshi, 1991, FEBS Lett. 281: 1-8),protoplast transformation via polyethylene glycol or electroporation(for review see Potyrkus, 1991, Annu. Rev. Plant Physiol. Plant Mol.Biol. 42: 205-225), microinjection of DNA into mesophyll protoplasts(Crossway et al., 1986, Mol. Gen. Genet. 202: 79-85), and macroinjectionof DNA into young floral tillers of cereal plants (de la Pena et al.,1987, Nature 325: 274-276) are preferred methods.

In general, the cDNA or gene encoding the phytase variant of theinvention is placed in an expression cassette (e.g., Pietrzak et al.,1986, Nucleic Acids Res. 14: 5857-5868) consisting of a suitablepromoter active in the target plant and a suitable terminator(termination of transcription). This cassette (of course including asuitable selection marker, see below) will be transformed into the plantas such in case of monocots via particle bombardment. In case of dicotsthe expression cassette is placed first into a suitable vector providingthe T-DNA borders and a suitable selection marker which in turn aretransformed into Agrobacterium tumefaciens. Dicots will be transformedvia the Agrobacterium harboring the expression cassette and selectionmarker flanked by T-DNA following standard protocols (e.g., Akama etal., 1992, Plant Cell Reports 12: 7-11). The transfer of T-DNA fromAgrobacterium to the Plant cell has been recently reviewed (Zupan &Zambryski, 1995, Plant Physiol. 107: 1041-1047). Vectors for planttransformation via Agrobacterium are commercially available or can beobtained from many labs that construct such vectors (e.g., Deblaere etal., 1985, Nucleic Acids Res. 13: 4777-4788; for review see Klee et al.,1987, Annu. Rev. Plant Physiol. 38: 467-486).

Available plant promoters: Depending on the process under manipulation,organ- and/or cell-specific expression as well as appropriatedevelopmental and environmental control may be required. For instance,it is desirable to express a phytase cDNA in maize endosperm etc. Themost commonly used promoter has been the constitutive 35S-CaMV promoterFranck et al., 1980, Cell 21: 285-294). Expression will be more or lessequal throughout the whole plant. This promoter has been usedsuccessfully to engineer herbicide- and pathogen-resistant plants (forreview see Stitt & Sonnewald, 1995, Annu. Rev. Plant Physiol. Plant Mol.Biol. 46: 341-368). Organ-specific promoters have been reported forstorage sink tissues such as seeds, potato tubers, and fruits (Edwards &Coruzzi, 1990, Annu. Rev. Genet. 24: 275-303), and for metabolic sinktissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24:863-878).

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed phytase may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous com-ponents of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

Preferred host cells are a strain of Fusarium, Hansenula, Trichoderinaor Aspergillus, in particular a strain of Fusarium graminearum, Fusariumvenenatum, Fusarium cerealis, Fusarium sp. having the identifyingcharacteristic of Fusarium ATCC 20334, as further described inPCT/US/95/07743, Hansenula polymorpha, Trichoderma harzianum orTrichoderma reesei, Aspergillus niger or Aspergillus oryzae.

References for expression in Hansenula polymorpha: Gellissen, G.,Piontek, M., Dahlems, U., Jenzelewski, V., Gavagan, J. E., DiCosimo, R.,Anton, D. I. & Janowicz, Z. A., 1996, Recombinant Hansenula polymorphaas a biocatalyst: coexpression of the spinach glycolate oxidase (GO) andthe S. cerevisiae catalase T (CTT1) gene. Appl. Microbiol. Biotechnol.46, 46-54.

Some more specific uses of the phytase variants according to theinvention appear from PCT/DK97/00568, the last pages of the detaileddescription of the invention section.

In a preferred embodiment, the phytase variant of the invention isessentially free of other non-phytase polypeptides, e.g., at least about20% pure, preferably at least about 40% pure, more preferably about 60%pure, even more preferably about 80% pure, most preferably about 90%pure, and even most preferably about 95% pure, as determined bySDS-PAGE. Sometimes such polypeptide is alternatively referred to as a“purified” and/or “isolated” phytase.

A phytase polypeptide which comprises a phytase variant of the inventionincludes fused polypeptides or cleavable fusion polypeptides in whichanother polypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof. A fused polypeptide is produced byfusing a nucleic acid sequence (or a portion thereof) encoding anotherpolypeptide to a nucleic acid sequence (or a portion thereof) encoding aphytase variant of the present invention. Techniques for producingfusion polypeptides are known in the art, and include, ligating thecoding sequences encoding the polypeptides so that they are in frame andthat expression of the fused polypeptide is under control of the samepromoter(s) and terminator.

A “feed” and a “food,” respectively, means any natural or artificialdiet, meal or the like or components of such meals intended or suitablefor being eaten, taken in, digested, by an animal and a human being,respectively.

The phytase variant of the invention may exert its effect in vitro or invivo, i.e., before intake or in the stomach of the individual,respectively. Also a combined action is possible.

A phytase composition according to the invention always comprises atleast one phytase of the invention.

Generally, phytase compositions are liquid or dry.

Liquid compositions need not contain anything more than the phytaseenzyme, preferably in a highly purified form. Usually, however, astabilizer such as glycerol, sorbitol or mono propylen glycol is alsoadded. The liquid composition may also comprise other additives, such assalts, sugars, preservatives, pH-adjusting agents, proteins, phytate (aphytase substrate). Typical liquid compositions are aqueous or oil-basedslurries. The liquid compositions can be added to a food or feed afteran optional pelleting thereof.

Dry compositions may be spray-dried compositions, in which case thecomposition need not contain anything more than the enzyme in a dryform. Usually, however, dry compositions are so-called granulates whichmay readily be mixed with e.g., food or feed components, or morepreferably, form a component of a pre-mix. The particle size of theenzyme granulates preferably is compatible with that of the othercomponents of the mixture. This provides a safe and convenient means ofincorporating enzymes into e.g., animal feed.

Agglomeration granulates are prepared using agglomeration technique in ahigh shear mixer (e.g., Lödige) during which a filler material and theenzyme are co-agglomerated to form granules. Absorption granulates areprepared by having cores of a carrier material to absorb/be coated bythe enzyme.

Typical filler materials are salts such as disodium sulphate. Otherfillers are kaolin, talc, magnesium aluminium silicate and cellulosefibers. Optionally, binders such as dextrins are also included inagglomeration granulates.

Typical carrier materials are starch, e.g., in the form of cassava,corn, potato, rice and wheat. Salts may also be used.

Optionally, the granulates are coated with a coating mixture. Suchmixture comprises coating agents, preferably hydrophobic coating agents,such as hydrogenated palm oil and beef tallow, and if desired otheradditives, such as calcium carbonate or kaolin.

Additionally, phytase compositions may contain other substituents suchas colouring agents, aroma compounds, stabilizers, vitamins, minerals,other feed or food enhancing enzymes, i.e., enzymes that enhance thenutritional properties of feed/food, etc. This is so in particular forthe so-called pre-mixes.

A “food or feed additive” is an essentially pure compound or a multicomponent composition intended for or suitable for being added to foodor feed. In particular it is a substance which by its intended use isbecoming a component of a food or feed product or affects anycharacteristics of a food or feed product. It is composed as indicatedfor phytase compositions above. A typical additive usually comprises oneor more compounds such as vitamins, minerals or feed enhancing enzymesand suitable carriers and/or excipients.

In a preferred embodiment, the phytase compositions of the inventionadditionally comprises an effective amount of one or more feed enhancingenzymes, in particular feed enhancing enzymes selected from the groupconsisting of alpha-galactosidases, beta-galactosidases, in particularlactases, other phytases, beta-glucanases, in particularbeta-1,4-endoglucanases and beta-1,3(4)-endoglucanases, cellulases,xylosidases, galactanases, in particular arabinogalactanbeta-1,4-endogalactosidases and arabinogalactanbeta-1,3-endogalactosidases, endoglucanases, in particularbeta-1,2-endoglucanase, alpha-1,3-endoglucanase, andbeta-1,3-endoglucanase, pectin degrading enzymes, in particularpectinases, pectinesterases, pectin lyases, polygalacturonases,arabinanases, rhamnogalacturonases, rhamnogalacturonan acetyl esterases,rhamnogalacturonan-alpha-rhamnosidase, pectate lyases, andalpha-galacturonisidases, mannanases, beta-mannosidases, mannan acetylesterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phospholipasesand cutinases.

The animal feed additive of the invention is supplemented to themono-gastric animal before or simultaneously with the diet. Preferably,the animal feed additive of the invention is supplemented to themono-gastric animal simultaneously with the diet. In a more preferredembodiment, the animal feed additive is added to the diet in the form ofa granulate or a stabilized liquid.

An effective amount of phytase in food or feed is from about 10-20.000;preferably from about 10 to 15.000, more preferably from about 10 to10.000, in particular from about 100 to 5.000, especially from about 100to about 2.000 FYT/kg feed or food.

Examples of other specific uses of the phytase of the invention are insoy processing and in the manufacture of inositol or derivativesthereof.

The invention also relates to a method for reducing phytate levels inanimal manure, wherein the animal is fed a feed comprising an effectiveamount of the phytase of the invention.

Also comprised in this invention is the use of a phytase of theinvention during the preparation of food or feed preparations oradditives, i.e., the phytase exerts its phytase activity during themanufacture only and is not active in the final food or feed product.This aspect is relevant for instance in dough making and baking.

The invention relates to a phytase variant which, when aligned accordingto FIG. 1, is amended as compared to a model phytase in at least one ofthe following positions, using the position numbering corresponding to P_(—) lycii:

24; 27; 31; 33; 39; 40; 41; 42; 43; 44; 45; 46; 47; 49; 51; 56; 58; 59;61; 62; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83;84; 88; 90; 102; 115; 116; 117; 118; 119; 120; 121; 122; 123; 124; 125;126; 127; 128; 132; 143; 148; 149; 150; 151; 152; 153; 154; 155; 156;157; 158; 159; 160; 161; 162; 163; 170f; 170g; 171; 172; 173; 184; 185;186; 187; 187a; 190; 191; 192; 193; 194; 195; 198; 199; 200; 201; 201a;201b; 201c; 201d; 201e; 201f; 202; 203; 203a; 204; 205; 211; 215; 220;223; 228; 232; 233; 234; 235; 236; 237; 238; 239; 242; 243; 244; 246;251e; 253; 256; 260; 264; 265; 267; 270; 271; 272; 273; 274; 275; 276;277; 278; 279; 280; 283; 285; 287; 288; 292; 293; 302; 304; 332; 333;334; 335; 336; 337; 338; 339; 340; 341; 342; 343; 348; 349; 352; 360;362; 364; 365; 366; 367; 368; 369; 370; 371; 372; 373; 374; 375; 376;383k; 387; 393; 394; 396; 404; 409; 411; 412; 413; 417; 421; 431.

From these variants we expect amended characteristics, preferablyamended activity characteristics. In fact, for several variants suchamended characteristics have already been shown (see the experimentalpart). Like above, “amended” means as compared to the model phytase.“Amended activity characteristics” means amended in at least one phytaseactivity related respect, such as (non-exclusive list): pH stability,temperature stability, pH profile, temperature profile, specificactivity (in particular in relation to pH and temperature), substratespecificity, substrate cleavage pattern, substrate binding, positionspecificity, the velocity and level of release of phosphate from corn,reaction rate, phytate degradation rate), end level of releasedphosphate reached.

Preferred amended activity characteristics are amended specificactivity, preferably increased, and preferably increased at a pH of 3,4, 5, or 6; amended pH or temperature profile; and/or amended,preferably increased, thermostability, e.g., of an increased meltingtemperature as measured using DSC.

Preferred phytase variants are: Phytase variants which, when alignedaccording to FIG. 1, are amended as compared to a model phytase in atleast one of the following positions, using the position numberingcorresponding to P _(—) lycii:

43; 44; 47; 51; 58; 62; 78; 80; 83; 88; 90; 102; 143; 148; 153; 154;186; 187a; 195; 198; 201e; 204; 205; 211; 215; 220; 242; 244; 251e; 260;264; 265; 267; 270; 273; 278; 302; 336; 337; 339; 352; 365; 373; 383k;404; 417.

The following variants of A _(—) fumigatus constitute a subgroup: Q43L;Q270L; G273D,K; N336S; A205E; Y278H; Q43L+Q270L; Q43L+Q270L+G273D;Q43L+Q270L+G273D+N336S; G273K+A205E; G273K+A205E+Y278H (see EP 0897010).

Generally, variants of the invention can be deduced or identified asfollows: Looking at the alignment according to FIG. 1, comparing twosequences, one of which is a model phytase with improved properties,identifying amino acid differences in relevant positions/areas, andtransferring (substituting with) from the model to the other phytasesequence the amino acid in a relevant position.

The invention also relates to a process for preparing a phytase variantwhich includes the above method, and further includes the deducement andsynthesis of the corresponding DNA sequence, the transformation of ahost cell, the cultivation of the host cell and the recovery of thephytase variant.

Relevant positions/areas include those mentioned below in relation toimportant phytase activity characteristics such as specific activity,thermostability, pH activity/stability.

The present invention also relates to phytase variants (varied accordingto a model phytase as defined herein) which are obtainable, preferablyobtained, by the process outlined above and which are expected toexhibit an amended characteristic/property, preferably does exhibit suchamended characteristic, e.g., an improved specific activity.

At least the basidiomycete model phytases P _(—) lycii and T _(—)pubescens exhibit a high specific activity (as determined using themethod of Example 2 herein).

This is an example of a desired property which can be transferred toother phytases, e.g., the other phytases listed in FIG. 1, in particularto the A _(—) pediades and the ascomycete phytases such as A _(—)fumigatus, A-ficuum, consphyA, by a deducement process such as the onementioned above.

Thus, amended specific activity, in particular an improved specificactivity, in particular at low pH and/or high temperature, is expectedfrom variants, which have been amended in relevant areas, viz. (i) inthe amino acid residues which point into the active site cleft; or (ii)in the amino acid residues in the close neighbourhood of these activesite residues. Preferably, close neighbourhood means within 10 Angstromsfrom the active site residues.

From the pdb file 1IHP (Brookhaven Database entry of 18.03.98 re 1IHP,Structure of Phosphomonoesterase, D. Kostrewa; or as published in NatureStructural Biology, 4, 1997, p. 185-190), active site regions can beidentified, using the program INSIGHTII from Molecular Simulations MSI,San Diego, Calif., and using the subset command, an “active site shell”can be defined comprising those amino acid residues which lie close tothe catalytic residues, defined as H59, D339 and R58 in A. ficuumphytase (corresponding to Peniophora numbers H71, D335 and R70,respectively). An “active site shell (10 Angstroms)” comprises thoseresidues which lie within 10 Angstroms from the above catalyticresidues.

The residues within 10 Angstroms from H71 and D335 are the following(using Peniophora numbers): 41-47, 68-77, 115-118, 120-126, 128,149-163, 185, 191-193, 199, 243, 270-271, 273-275, 277-279, 288,332-343, 364-367, 369-375, 394 (“the active site shell (10 Angstroms)”).

Preferably, a “substrate binding shell” can also be defined whichcomprises those residues which are in close proximity to the substratebinding site and which can therefore be expected to be in contact withthe substrate.

This information can be deduced as described above, by docking a sugaranalogue to phytin into the active site cleft (the residues making upthe surface of the active site). If a sugar without any phosphate groupsis docked into the active site cleft, e.g., alpha-D-glucose (chairconformation, structure provided by the INSIGHTII program), using afixed distance as shown below, the residues pointing towards the activesite cleft can be extracted using the subset command and using adistance of 10 Angstroms from the substrate analogue. Alternatively, thecompound inositol-1,4,5-triphosphate (Brookhaven database file 1djx.Inositol-1,4,5-triphosphate) can be docked into the active site cleft.This compound and glucose, however, are more or less superimposable.

The distances in Angstroms are: From oxygen atom in position 6 of thealpha-D-glucose to

atom ND1 of H59: 5.84 atom NH2 of R58: 6.77 atom NH2 of R142: 5.09 atomND2 of N340: 3.00 atom ND1 of H59: 7.76 atom NH2 of R58: 8.58(the Peniophora numbers of the above residues are: H71, R70, R155, N336,H71 and R70, respectively).

In this way, the residues in contact with the substrate are identifiedas follows (Peniophora numbers): 43-44; 70-80; 83-84; 115; 153; 155-156;184; 191-192; 198-202; 205; 235; 238; 242; 270; 272-273; 275-277;332-336; 338; 369; 371 (“the substrate binding shell (10 Angstroms)”).

Variants being amended in one or more of (1) the active site shell or(2) the substrate binding shell are strongly expected to have an amendedspecific activity. This leads to the following joint grouping ofpositions (still Peniophora numbers and 10 Angstrom shells): 41-47,68-80, 83-84, 115-118, 120-126, 128, 149-163, 184-185, 191-193,198-201e, 202-203, 205, 235-236, 238-239, 242-243, 270-279, 285, 288,332-343, 364-367, 369-375, 394.

Preferably, the active site shell and the substrate binding shell aredefined as described above using the basidiomycete model phytases ofFIG. 1, the Peniophora phytase being a preferred model. A deducement ofcorresponding variants of other model phytases is possible using thealignment of FIG. 1.

In a preferred embodiment, a distance of 5 Angstroms is used in thesubset command, thus defining active site and substrate binding shellsof a more limited size, e.g., an active site shell comprising theresidues 43-44, 69-74, 117, 125, 155-156, 159, 274, 332-340, 370-374 (5Angstroms from H71 and D335), “active site shell (5 Angstroms)”.

Generally the active site shell and substrate binding shell regions formthe basis for selecting random mutagenesis regions. Examples ofpreferred random mutagenesis regions are

regions 69-74, 332-340, 370-374, doping to be added (a 5 Angstromapproach); and

regions 57-62, 142-146, 337-343, doping to be added (a 10 Angstromapproach).

It is presently contemplated that any amendment in either of thesepositions will lead to a phytase of amended characteristics, e.g., of anamended specific activity.

The above expression “any amendment in either of the positions” isconsidered fully equivalent to listing each position and eachsubstitution, e.g., as follows for the above sub-group 41-47:

41A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;42A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;43A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;44A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;45A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;46A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;47A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y.

In a preferred embodiment, amended specific activity is expected fromthe following variants:

42S,G; 43A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 45D,S; 47Y,F; 51E,A;75W,F; 78S,D; 79G; 80K,A; 83I,Q; 84Q,V; 116S; 118V,L; 119E; 120L; 122A;123N,T; 125S; 126H,S; 127Q,E; 128A,T; 151A,S; 152G; 153D,Y; 154Q,D,G;157V; 158D,A; 159T; 160A,S; 161T,N; 162N; 163W; 184Q,S; 186A,E; 198A,N;200G,V; 201D; deletions of one or more of 201a, 201b, 201c, 201d, 201e,201f—preferably all; 202S; 205Q,E; 235Y,L; 238L,M; 242P; 270Y,A,L; 271D;273D,K; 275F,Y; 278T,H; 332F; 336S; 337T,Q; 339V; 340P,A; 343A,S;364W,F; 365V,L; 366D,V; 367K; 368K; 369I,L; 370V; 373S; 374A; 375H;376M; 393V.

Particularly preferred variants are the following: 78S; 79G; 80A; 83I,Q;84Q,V; 198A,N; 200G,V; 201D; deletions in one or more of 201a, 201b,201c, 201d, 201e, 201f—preferably all deletions; 202S; 205Q,E; 235Y,L;238L,M; 242P, 273D; 275F,Y.

Other particularly preferred variants are the following:43A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; in particular 43M,P; 75W,F;80K; 153D; 184Q,S; 270Y,A; 332F; 369I,L.

The following variants are especially preferred: 43L,G,N,V,A,I,T; 78D;153Y; 154G; 270L; 273D,K. Double and triple variants (43L/270L);(43L/270L/273D); (43L/78D) and (43L/153Y/154G) are also especiallypreferred. Other preferred variants are 205E; 278H; 336s.

These especially preferred single, double and triple variants arepreferably variants of model phytases which can be aligned to FIG. 1, inparticular variants of the specific model phytases listed in FIG. 1.

At least consphyA is known to have a high thermostability. Stillfurther, the thermostability of P _(—) lycii is rather high.

This is an example of a desired property which can be transferred toother phytases, e.g., the other phytases listed in FIG. 1, in particularto the basidiomycete phytases such as P _(—) lycii and A _(—) pediades,by a deducement process such as the one mentioned above.

Amended thermostability, in particular improved thermostability, isexpected on this background from the following variants:

39H,S; 40L,N; 43P; 47Y,F; 49P; 51E,A; 56P; 58D; 61R; 62V; 80K; 83A; 84Y;172P; 184P; 195T; 198A; 204V; 211L; 223D; 236Y; 242P; 246V; 253P; 264R;265Q; 280A,P; 283P; 287A; 292F,Y; 293A; 302R; 304P; 337S; 348Y; 387P;396R; 409R; 411K; 412R; 417E; 421F,Y.

The following variants of amended thermostability are particularlypreferred: 39S; 40N; 47Y,F; 51A; 83A; 195T; 204V; 211L; 242P; 265A.

Further variants of amended thermostability are the following: 42G;43T,L,G; 44N; 58K,A; 59G; 62I; 69Q; 75F; 78D; 79G; 80A; 81A,G; 82T;83K,R; 84I; 88I; 90R,A; 102Y; 115N; 118V; 122A; 123Q,N; 125M,S; 126V,S;127N,Q; 128S,A; 143N,K; 148V,I; 154S; 158D; 170fH; 170gA; 171T,N; 172N;173W, 184S; 186A; 187A; 187aS; 193S; 195V,L; 198V; 201E; 201eT; 202A,203aT; 204A; 211V, 215P,A; 220L,N; 223H; 228N; 232T; 322E; 235T; 236N;242s; 244D; 251eQ,E; 256D; 264I; 260A,H; 265A; 267D; 270G; 271D; 273K,D;278T,H; 287T; 293V; 302H; 337T,G; 338I; 339V,I; 340A; 352K; 365A,S;366S; 367A; 369L; 373S,A; 374S; 376M; 383kE,Q; 404G,A; 411T; 417R; 431E.

Other concepts of the invention, which can be expected to impart animproved thermostability to a phytase, are as follows—considering the1IHP structure previously referred to and transferring via an alignmentaccording to FIG. 1 as outlined herein:

(A) Introduction of proline residues in spatial positions where theprolin special dihedral angles are satisfied and the hydrogen bondingnetwork are not hampered and no steric clashes are observed.(B) Filling up holes: By substitution for bigger residues in internalcavities an improvement in stability can often be obtained.(C) Cystin bridge: Cystin bridges will often make the proteins morerigid and increase the energy of unfolding.

Further variants from which amended thermostability is expectedaccording to these concepts of (A) to (C) are: 27P, 31Y, 132F, 132I,132L, 184P, 186P, 190P, 280P, 343F, 343I, 343L, 349P, 362P and (33C and24C).

Concept (A): 27P, 184P, 186P, 190P, 349P, 362P.

Concept (B): 343F,I,L; 31Y; 132F,I,L; 273F.

Concept (C): 33C/24C.

Amended pH activity or stability, preferably stability, in particular atlow pH, in particular improved, is another desired property which can betransferred by aligning according to FIG. 1 and transferring from modelsof improved pH profiles to other phytases—as outlined above.

Other concepts of the invention, which can be expected to impart animproved stability at low pH to a phytase, are as follows—consideringthe 1IHP structure previously referred to and transferring via analignment according to FIG. 1 as outlined herein:

(D) Surface charges: Better distribution at low pH, to avoid cluster ofnegative or positive, and to avoid too close same charged residues.(E) Prevent deamidation: Surface exposed Q or N in close contact tonegative charged residues.

Phytase variants having improved pH stability/activity at low pH areexpected to be: 39H; 39Q; 80A; 203R; 271N; 51R; 154S; 185S; 194S; 194T;288L; 288I; 288F; 360R; 173Q,S; 204Q,S; 303K,S; 81Q,E.

Concept (D): 203R, 271N, 51R, 185S, 360R; 173Q,S; 204Q,S; 303K,S; 81Q,E.

Concept (E): 154S; 194S,T; 288L,I,F.

A preferred model phytase for these concepts of (D) and (E) is P _(—)lycii.

Experimentally proven to have a lowered pH optimum is: Variant 80A ofascomycete phytases, in particular of A _(—) fumigatus and consphyA.

Especially preferred single, double and triple variants are 43L;(43L/270L) and (43L/270L/273D). These variants have a changed pHprofile. They are preferably variants of the specific model phytaseslisted in FIG. 1.

For all preferred variants listed above:

the stability is preferably amended at high temperature, viz. in thetemperature range of 50-100° C., in particular 60-90° C., morepreferably in the range of 70-90° C.;

the activity is preferably amended in a temperature range relevant forthe use in the gastro-intestinal system of animals, e.g., 30-40° C.,more preferably 32-38° C., most preferably in the range of 35-38° C.;

the stability is preferably amended at low pH, viz. in the pH range ofpH 1.5-7, preferably 2-6, more preferably 3-5;

the activity is preferably amended in the pH range of pH 1.5-5.5, morepreferably at pH 2.5-4.5, still more preferably 3-5.

Tests for amended phytase characteristics, such as those mentionedabove, are well known in the art and any such test can be used tocompare the performance of the phytase variants with the phytase models.

A preferred test for specific activity is given in Example 2. Preferredtests for pH and temperature activity and stability are given in Example3. An even more preferred test for thermal stability is the DSC methodof Example 4.

WO 98/28409 discloses tests for various other parameters, too, such asposition specificity. All the tests of WO 98/28409 are preferred tests.

Generally, of course all these tests can be conducted at desired pHvalues and temperatures.

In the dependent claims, some preferred phytase variants based on fiveof the thirteen herein specifically disclosed model phytases arespecified.

In an analogous way other preferred variants based on the remainingeight specifically disclosed model phytases can easily be deduced bycombining the suggested amendments with each of the correspondingsequences of FIG. 1. These preferred variants are specifically includedin the present invention, and they are easily deduced, viz. thefollowing:

Variants of a model phytase derived from Paxillus, preferably Paxillusinvolutus, preferably derived from strain CBS 100231, preferablyvariants of P _(—) involtus-A1, the sequence of which is shown at FIG.2, said variants comprising at least one of the following amendments:

( )24C; T27P; F31Y; I33C; R39H,S,Q; N40L; S42G;P43A,C,D,E,F,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y; Y44N; S45D; Y47F; A51E,R;A58D;K; Q61R; I62V; F75W; S78D; A80K; T81Q,E,G,A; R83A,I,Q,K; I84Y,Q,V;L88I; K90R,A; F102Y; S115N; D116S; V118L; P119E; F120L; A123N,T,Q;S125M; F126H,S,V; D127Q,E,N; A128T,S; A132F,I,L; I148V; D151A,S;S153D,Y; D154Q,S,G; D158A; S159T; A160S; T161N; ( )170fH; ( )170gA;S171N; H172P; N173Q,S; P184Q,S; Q185S; T186A,E,P; G187A; ( )187aS;T190P,A; D193S; N194S,T; M195T,V,L; A198N,V; G200V; D201E; ( )201eT;S202A; D203R,K,S; P203aV,T; Q204E,S,A,V; V205E; V211L; S215A,I; L220N;A223D,H; D233E; F235Y,L,T; N236Y; L237F; V238L,M; A242P,S; M244D; ()251eE,Q; D253P; T256D; P260A,H; E264R,I; A265Q; A267D; G270Y,A,L;D271N; D273K; F275Y; T278H; Y280A,P; E283P; V287A,T; Q288L,I,F; Y292F;V293A; N₃O₂R,H; A304P; N336S; L337T,Q,S,G; M 338I; V339I; A340P;S343A,F,I,L; F348Y; R349P; A352K; P360R; R362P; W364F; R365V,L,A,S;T366D,V,S; S367K,A; S368K; L369I; S373A; G374A,S; R375H; ( )383kQ,E;T387P; Q396R; G404A; L409R; T411K; L412R; E417R; F421Y.

Variants of a model phytase derived from a species of the genusPaxillus, preferably the species Paxillus involutus, preferably derivedfrom strain CBS 100231, preferably variants of P _(—) involtus-A2, thesequence of which is shown at FIG. 3, said variants comprising at leastone of the following amendments:

P24C; I27P; F31Y; I33C; R39H,S,Q; N40L; S42G;P43A,C,D,E,F,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y; Y44N; S45D; Y47F; A51E,R;A58D,K; E61R; I62V; F75W; S78D; A80K; A81Q,E,G; R83A,I,Q,R,K; I84Y,Q,V;L88I; K90R,A; F102Y; S115N; D116S; V118L; P119E; F120L; A123N,T,Q;S125M; F126H,S,V; D127Q,E,N; A128T,S; V132F,I,L; D143N; I148V; D151A,S;S153D,Y; D154Q,S,G; D158A; A160S; T161N; ( )170fH; ( )170gA; S171N;R172P; N173Q,S; P184Q,S; Q185S; T186A,E,P; G187A; ( )187aS; T190P,A;D193S; N194S,T; M195T,V,L; A198N,V; G200V; E201D; ( )201eT; S202A;D203R,K,S; P203aV,T; Q204E,S,A,V; V205E; S211L,V; S215A,P; L220N;A223D,H; A232T; F235Y,L,T; N236Y; L237F; V238L,M; P242S; M244D; ()251eE,Q; D253P; T256D; P260A,H; E264R,I; A265Q; A267D; G270Y,A,L;D271N; D273K; F275Y; T278H; Y280A,P; A283P; V287A,T; Q288L,I,F; Y292F;I293A,V; N₃O₂R,H; A304P; N336S; L337T,Q,S,G; M338I; V339I; 340P,A;A343S,F,I,L; F348Y; R349P; A352K; P360R; R362P; W364F; L365V,A,S;T366D,V,S; S367K,A; S368K; V369I,L; S373A; R375H; ( )383kQ,E; T387P;Q396R; G404A; L409R; A411K,T; L412R; E417R; Y421F.

Variants of a model phytase derived from a species of the genusTrametes, preferably the species Trametes pubescens, preferably derivedfrom strain CBS 100232, preferably variants of T _(—) pubescens, thesequence of which is shown at FIG. 4, said variants comprising at leastone of the following amendments:

R24c; T27P; L31Y; V33C; Q39H,S; S40L,N; S42G;M43A,C,D,E,F,G,H,I,K,L,N,P,Q,R,S,T,V,W,Y; Y44N; S45D; Y47F; A51E,R;A58D,K; S59G; Q61R; I62V; F75W; S78D; A80K; A81Q,E,G; R83A,I,Q,K;I84Y,Q,V; V88I; K90R,A; L102Y; D115N; V118L; T123N,Q; S125M; S126H,V;E127Q,N; A128T,S; A132F,I,L; D143N; V148I; S151A; S153D,Y; D154Q,S,G;A158D; A160S; N161T; ( )170fH; ( )170gA; S171N; S172P; N173Q,S; S184Q,P;E185S; A186E,P; G187A; ( )187aS; T190P,A; N194S,T; M195T,V,L; A198N,V;G200V; ( )201eT; S202A; D203R,K,S; P203aV,T; Q204E,S,A,V; V205E;Q211L,V; P215A; L220N; G223D,H; D233E; Y235L,T; N236Y; L237F; L238M;P242S; E244D; ( )251eE,Q; E253P; Q260A,H; D264R,I; A265Q; A267D;A270Y,L,G; D271N; D273K; F275Y; T278H; Y280A,P; V287A,T; Q288L,I,F;Y292F; I293A,V; A302R,H; N₃O₄P,A; N336S; Q337T,S,G; M338I; V339I; A340P;S343A,F,I,L; F348Y; N349P; A352K; P360R; R362P; F364W; L365V,A,S;V366D,S; K367A; I369L; A373S; A374S; R375H; ( )383kQ,E; Q387P; A396R;G404A; V409R; T411K; L412R; E417R; Y421F.

Variants of a model phytase derived from a species of the genusAspergillus, preferably the species Aspergillus nidulans, preferablyderived from strain DSM 9743, preferably variants of A _(—) nidulans,the sequence of which is shown at FIG. 10, said variants comprising atleast one of the following amendments:

V24C; A27P; H39S,Q; V40L,N; G42SQ43A,C,D,E,F,G,H,I,K,L,M,N,P,R,S,T,V,W,Y; Y44N; S45D; Y47F; S49P;E51A,R; V56P; H58D,K,A; E61R; V62I; S69Q; Y75W,F; E78D,S; S79G; K80A;S81Q,E,A,G; K82T; A83I,Q,K,R; Y84Q,V,I; A90R; D115N; D116S; T118V,L;I119E; F120L; E122A; N123T,Q; M125S; V126H,S; D127Q,E,N; S128A,T;F132I,L; K143N; I148V; S151A; S153D,Y; D154Q,S,G; A158D; S159T; A160S;E161T,N; K162N; F163W; G170fH; S170gA; ( )171N; ( )172P; K173Q,S;P184Q,S; E185S; I186A,E,P; D187A; G187aS; T190P,A; H193S; S194T;S198A,N,V; E200G,V; N201D,E; D201e( ); E201e ( ), T; R201f ( ) (adeletion of at least one of 201d, 201e, 201f, preferably all); A202S;D203R,K,S; E203aV,T; I204Q,E,S,A,V; I211L,V; P215A; L220N; D223H; K228N;E232T; N233E; I235Y,L,T; Y236N; L237F; M238L; S242P; M246V; E251eQ;A256D; E260A,H; L264R,I; Q270Y,A,L,G; S271D,N; S273D,K; Y275F; G278T,H;A280P; A287T; Q288L,I,F; F292Y; T293A,V; Q302R,H; P304A; N336S;S337T,Q,G; M338I; I339V; S340P,A; F343A,S,I,L; N349P; Q352K; S360R;Q362P; Y364W,F; A365V,L,S; A366D,V,S; S367K,A; W368K; T369I,L; G373S,A;A374S; R375H; A376M; E383kQ; A404G; T411K; L412R; E417R; F421Y; K431E.

Variants of a model phytase derived from a species of Aspergillus,preferably Aspergillus terreus, preferably derived from strain CBS220.95, preferably variants of A _(—) terreus, the sequence of which isshown at FIG. 12, said variants comprising at least one of the followingamendments:

G24C; V27P; H39S,Q; K40L,N; G42S;L43A,C,D,E,F,G,H,I,K,M,N,P,Q,R,S,T,V,W,Y; Y44N; A45D,S; Y47F; S49P;Q51E,A,R; V56P; P58D,K,A; D59G; H61R; I62V; A69Q; S75W,F; H78D,S; S79G;K80A; T81Q,E,A,G; A83I,Q,K,R; Y84Q,V,I; A90R; E115N; E116S; T118V,L;P119E; F120L; R122A; N123T,Q; L125S,H; R126H,S,V; D127Q,E,N; L128A,T,S;F132I,L; H143N; V148I; T151A,S; D152G; A153D,Y; S154D,Q,G; H157V;E158D,A; S159T; A160S; E161T,N; K162N; F163W; H173Q,S; P184Q,S; E185S;G186A,E,P; S187A; A187aS; T190P,A; H193S; S194T; L195T,V; A198N,V;E200G,V; S201D,E; S201d( ); T201e( ); V201f( ); G202S,A; D203R,K,S;D203aV,T; A204Q,E,S,V; V205E; V211L; A215P; L220N; D223H; Q228N; D232T;D233E; V235Y,L,T; N236Y; L237F; M238L; P242S; E244E; T251eE,Q; A260H;T264R,I; Q265A; N267D; L270Y,A,G; S271D,N; K273D; Y275F; H278T; G280A,P;V287A,T; Q288L,I,F; W292F,Y; A293V; Q302H; P304A; N337T,Q,S,G; L338I;V339I; S340P,A; W343A,S,F,I,L; N349P; A352K; S360R; S362P; Y364W,F;A365V,L,S; A366D,V,S; A367K; W368K; T369I,L; A373S; A374S; R375H; A376M;R383kQ,E; P404A,G; K411T; A417E,R; F421Y; A431E.

Variants of a model phytase derived from a species of Talaromyces,preferably the species Talaromyces thermophilus, preferably derived fromstrain ATCC 20186 or ATCC 74338, preferably variants of T _(—) thermo,the sequence of which is shown at FIG. 13, said variants comprising atleast one of the following amendments:

H₂₄C; V27P; H39S,Q; S40L,N; G42S;Q43A,C,D,E,F,G,H,I,K,L,M,N,P,R,S,T,V,W,Y; Y44N; S45D; F47Y; S49P;A51E,R; V56P; Q58D,K,A; N59G; K61R; I62V; Y75W,F; S78D; S79G; K80A;T81Q,E,A,G; E82T; L83A,I,Q,R,K; Y84Q,V,I; R90A; D116S; T118V,L; P119E;F120L; E122A; N123T,Q; M125S; I126H,S,V; Q127E,N; L128A,T,S; F132I,L;V148I; S151A; S153D,Y; D154Q,S,G; I157V; A158D; S159T; G160A,S; R161T,N;L162N; F163W; S170gA; D171N; K172P; H173Q,S; E184Q,S,P; E185S;G186A,E,P; D187A; T190P,A; T1933; G194S,T; S195T,V,L; V198A,N; E200G,V;D201E; S201d( ); S201e( ),T; S201f( ); G202S,A; H203R,K,S; D203aV,T;A204Q,E,S,V; Q205E; Q211L,V; A215P; I220N,L; H223D; D228N; S232T; D233E;P235Y,L,T; Y236N; M237F; D238L,M; P242S; E244D; L246V; ( )251eE,Q;A256D; Q260A,H; Q264R,I; A265Q; Q270Y,A,L,G; S271D,N; G273D,K; Y275F;N278T,H; G280A,P; A287T; Q288L,I,F; F292Y; V293A; H302R; P304A; N336S;T337Q,S,G; M338I; T339V,I; S340P,A; A343S,F,I,L; N349P; A352K; S360R;E362P; Y364W,F; S365V,L,A; A366D,V,S; A367K; W368K; T369I,L; G373S,A;G374A,S; R375H; A376M; D383kQ,E; E404A; K411T; R417E; F421Y.

Variants of a model phytase derived from a species of Thermomyces,preferably the species Thermomyces lanuginosus, preferably derived fromstrain DBS 586.94, preferably variants of T _(—) lanuginosa, thesequence of which is shown at FIG. 14, said variants comprising at leastone of the following amendments:

K24C; ( )27P; ( )31Y; ( )33C; R39H,S,Q; H40L,N; G42S;Q43A,C,D,E,F,G,H,I,K,L,M,N,P,R,S,T,V,W,Y; Y44N; S45D; F47Y; S49P;A51E,R; V56P; K58D,A; V62I; S69Q; Y75W,F; A78D,S; H79G; K80A;S81Q,E,A,G; E82T; V83A,I,Q,K,R; Y84Q,V,I; L88I; R90A; F102Y; D115N;N116S; T118V,L; R119E; F120L; E122A; E123N,T,Q; M125S; M126H,S,V;E127Q,N; S128A,T; F132I,L; E143N; V148I; A151S; S153D,Y; A154D,Q,S,G;I157V; A158D; S159T; A160S; E161T,N; F162N; F163W; R170fH; S170gA;K172P; D173Q,S; S184Q,P; E185S; E186A,P; T187A; G187aS; T190P,A; G193S;L194S,T; T195V,L; A198N,V; E200G,V; E201D; A201d( ); P201e( ),T;D202S,A; P203R,K,S; T203aV; Q204E,S,A,V; P205E; V211L; R215A,P; I220L,N;H223D; E232T; D233E; P235Y,L,T; L236Y,N; M238L; P242S; Q251eE; H256D;Q260H; M264R,I; A265Q; Y270A,L,G; T271D,N; D273K; Y275F; H278T; G280A,P;A283P; S287A; R288L,I,F; F292Y; V293A; G302R,H; P304A; N336S; T337Q,S,G;M338I; T339V,I; G340P,A; S343A,F,I,L; N349P; P360R; T362P; Y364W,F;A365V,L,S; A366D,V,S; S367K,A; W368K; T369I,L; A373S; A374S; R375H;A376M; E383kQ; R404A,G; R411K,T; K417E,R; F421Y; D431E.

Variants of a model phytase derived from a species of Myceliophthora,preferably the species Myceliophthora thermophila, preferably derivedfrom strain ATCC 48102 or ATCC 74340, preferably variants of M _(—)thermophila, the sequence of which is shown at FIG. 7, said variantscomprising at least one of the following amendments:

S24C; F31Y; H39S,Q; F40L,N; G42S;Q43A,C,D,E,F,G,H,I,K,L,M,N,P,R,S,T,V,W,Y; Y44N; S45D; Y47F; S49P;P51E,A,R; I56P; D58K,A; D59G; E61R; V62I; S69Q; A75W,F; L78D,S; K79G;R80K,A; A81Q,E,G; A82T; S83A,I,Q,K,R; Y84Q,V,I; R90A; D115N; E116S;T118V,L; R119E; T120L; Q122A; Q123N,T; M125S; V126H,S; N127Q,E; S128A,T;F132I,L; K143N; V148I; A151S; Q153D,Y; D154Q,S,G; H158D,A; S159T; A160S;E161T,N; G170fH; S170gA; T171N; F163W; V172P; R173Q,S; P184Q,S; E185S;T186A,E,P; G187aS; T190P,A; N193S; D194S,T; L195T,V; A198N,V; E200G,V;E201D; G201a( ); P201b( ); Y201c( ); S201d( ); T201e( ); I201f( );G202S,A; D203R,K,S; D203aV,T; A204Q,E,S,V; Q205E; T211L,V; P215A;V220N,L; N223D,H; A232T; D233E; V235Y,L,T; A236Y,N; L237F; M238L; P242S;E244D; A251eE,Q; R256D; E260A,H; R264I; A265Q; Q270Y,A,L,G; S271D,N;K273D; Y275F; Y278T,H; P280A; T287A; Q288L,I,F; F292Y; V293A; ( )302R,H;P304A; N336S; D337T,Q,S,G; M338I; M339V,I; G340P,A; G343A,S,F,I,L;D349P; P352K; D360R; E362P; Y364W,F; A365V,L,S; A366D,V,S; S367K,A;W368K; A369I,L; A373S; A374S; R375H; I376M; E383kQ; E387P; G404A; M409R;T411K; L412R; E417R; F421Y; D431E.

This invention also provides a new phytase which has been derived from astrain of Cladorrhinum, viz. C. foecundissimun. Accordingly, theinvention also relates to a polypeptide having phytase activity andwhich comprises SEQ ID NO: 2 or the mature part (amino acids nos 16-495)thereof; or a polypeptide being at least 70, more preferably 75, 80, 85,90, 95% homologous thereto; homology meaning similarity, preferablyidentity, and being determined using the program GAP and the settings asdefined hereinabove. And the invention relates to a DNA construct whichencodes a polypeptide having phytase activity, said DNA constructcomprising a DNA molecule which comprises SEQ ID NO: 1 or nucleotidesnos. 20-70 and 207-1560 thereof; or nucleotides nos. 20-70 and 207-1563thereof; or nucleotides nos. 65-70 and 207-1560 thereof; or nucleotidesnos. 65-70 and 207-1563 thereof; or a DNA construct or molecule which isat least 70, 75, 80, 85, 90, 95% homologous to either of thesenucleotide sequences; homology meaning similarity, preferably identity,and being determined using computer programs known in the art such asGAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1996, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch,C. D., (1970), Journal of Molecular Biology, 48, 443-453). Using GAPwith the following settings for DNA sequence comparison: GAP creationpenalty of 5.0 and GAP extension penalty of 0.3. The invention alsorelates to a DNA construct which hybridizes with any of the above DNAsequences under the conditions mentioned hereinabove.

EXAMPLES Example 1 Phytase Activity Assay (FYT)

Phytase activity can be measured using the following assay: 10microliter diluted enzyme samples (diluted in 0.1 M sodium acetate,0.01% Tween20, pH 5.5) are added into 250 microliters 5 mM sodiumphytate (Sigma) in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5 (pHadjusted after dissolving the sodium phytate; the substrate ispreheated) and incubated for 30 minutes at 37° C. The reaction isstopped by adding 250 microliters 10% TCA and free phosphate is measuredby adding 500 microliters 7.3 g FeSO₄ in 100 ml molybdate reagent (2.5 g(NH₄)₆Mo₇O₂₄.4H₂0 in 8 ml H₂SO₄ diluted to 250 ml). The absorbance at750 nm is measured on 200 microliter samples in 96 well microtiterplates. Substrate and enzyme blanks are included. A phosphate standardcurve is also included (0-2 mM phosphate). 1 FYT equals the amount ofenzyme that releases 1 micromol phosphate/min at the given conditions.

Example 2 Test for Specific Activity

The specific activity can be determined as follows:

A highly purified sample of the phytase is used (the purity is checkedbeforehand on an SDS poly acryl amide gel showing the presence of onlyone component).

The protein concentration in the phytase sample is determined by aminoacid analysis as follows: An aliquot of the phytase sample is hydrolyzedin 6 N HCl, 0.1% phenol for 16 h at 110° C. in an evacuated glass tube.The resulting amino acids are quantified using an Applied Biosystems420A amino acid analysis system operated according to the manufacturer'sinstructions. From the amounts of the amino acids the total mass—andthus also the concentration—of protein in the hydrolyzed aliquot can becalculated.

The activity is determined in the units of FYT. One FYT equals theamount of enzyme that liberates 1 micromol inorganic phosphate fromphytate (5 mM phytate) per minute at pH 5.5, 37° C.; assay describede.g., in example 1.

The specific activity is the value of FYT/mg enzyme protein.

Example 3 Test for Temperature and pH Activity and Stability

Temperature and pH activity and stability can be determined as follows:

Temperature profiles (i.e., temperature activity relationship) byrunning the FYT assay of Example 1 at various temperatures (preheatingthe substrate).

Temperature stability by pre-incubating the phytase in 0.1 M sodiumphosphate, pH 5.5 at various temperatures before measuring the residualactivity.

The pH-stability by incubating the enzyme at pH 3 (25 mM glycine-HCl),pH 4-5 (25 mM sodium acetate), pH 6 (25 mM MES), pH 7-9 (25 mM Tris-HCl)for 1 hour at 40° C., before measuring the residual activity.

The pH-profiles (i.e., pH activity relationship) by running the assay atthe various pH using the same buffer-systems (50 mM, pH re-adjusted whendissolving the substrate).

Example 4 DSC as a Preferred Test for Thermostability

The thermostability or melting temperature, Tm, can be determined asfollows:

In DSC the heat consumed to keep a constant temperature increase in thesample-cell is measured relative to a reference cell. A constant heatingrate is kept (e.g., 90° C./hour). An endo-thermal process (heatconsuming process—e.g., the unfolding of an enzyme/protein) is observedas an increase in the heat transferred to the cell in order to keep theconstant temperature increase.

DSC can be performed using the MC2-apparatus from MicroCal. Cells areequilibrated 20 minutes at 20° C. before scanning to 90° C. at a scanrate of 90°/h. Samples of e.g., around 2.5 mg/ml phytase in 0.1 M sodiumacetate, pH 5.5 are loaded.

Example 5 Phytase Variants of Amended Activity Characteristics

Variants of an Aspergillus fumigatus model phytase (a wild type phytasederived from strain ATCC 13073) were prepared as described in EP98104858.0 (EP-A-0897010), examples 2-3 and 5, and the phytase activitywas determined as described in example 7 thereof. pH and temperatureoptima and melting point were determined as described in examples 9 and10 of EP 98113176.6 (EP-A-0897985).

In Table 1, variants of improved specific activity at pH 5.0 are listed.Table 2 lists variants of improved relative activity at pH 3.0, andTable 3 lists variants of improved thermostability (temperature optimum,e.g., determined by DSC).

TABLE 1 Amended in position Specific activity at no. Substitution intopH 5.0 (U/mg) 43 43L 83.4 43N 45.5 43T 106.9 43I 91.2 43V 35.0 43A 27.343G 59.6 43 and 270 43L, 270L 88.7 43 and 270 and 273 43L, 270L, 273D92.3 43 and 78 43L, 78D 118.5 43 and 153 and 154 43L, 153Y, 154G 193.0A. fumigatus wild-type — 26.5 phytase

TABLE 2 Amended in position Relative phytase no. Substitution intoactivity at pH 3.0 205 205E 41% 273 273K 61% 278 278H 75% 273 and 205273K, 205E 65% 273 and 278 273K, 278H 100% 273 and 205 and 278 273K,205E, 278H 96% A. fumigatus wild-type — 32% phytase

TABLE 3 Temperature Amended in position optimum Tm (° C.) no.Substitution into (° C.) (DSC) 43 and 47 and 88 and 43T, 47Y, 88I, 102Y,60 67 102 and 220 and 242 220L, 242P, 267D and 267 as above plus 51 andas above plus 51A, 63 — 302 and 337 and 373 302H, 337T, 373A, and 115115N A. fumigatus wild-type — 55 62.5 phytase

Example 6 Further Phytase Variants of Amended Activity Characteristics

Variants of the ascomycete consensus sequence “conphys” of FIG. 9 wereprepared as described in EP 98113176.6 (EP-A-0897985), examples 4-8.Phytase activity, including pH- and temperature optimum, and meltingpoint was determined as described in examples 9 and 10, respectively,thereof.

The tables below list variants of amended activity characteristics, viz.

Table 4 variants of improved specific activity at pH 6.0;

Table 5 variants of amended pH optimum (the pH-optimum indicated is anapproximate value, determined as that pH-value (selected from the groupconsisting of pH 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; and 7.0) at which themaximum phytase activity was obtained);

Table 6 a variant of improved thermostability (expressed by way of themelting point as determined by differential scanning calorimetry (DSC));and

Table 7 variants of amended thermostability (temperature optimum); a “+”or “−” indicates a positive or a negative, respectively, effect ontemperature optimum of up to 1° C.; and a “++” and “− −” means apositive or a negative, respectively, effect on temperature optimum ofbetween 1 and 3° C.

TABLE 4 Amended in position Specific activity at no. Substitution intopH 6.0 (U/mg) 43 43T 130 43L 205 Conphys — 62

TABLE 5 Amended in position pH optimum no. Substitution into around 4343T 6.0 43L 5.5 43G 6.5 43 and 44 43L, 44N 6.0 43T, 44N 5.5 Conphys —6.0

TABLE 6 Amended in Substitution position no. into Tm (° C.) 43 43T 78.9Conphys — 78.1

TABLE 7 Amended in Substitution Temperature optimum position no. intoamendment  51 A +  58 K + 220 N + 195 L ++ 201e T ++ 244 D + 264 I + 302H + 337 T ++ 352 K + 373 A ++  47 F −  62 I −  83 K −  90 R − 143 N −148 V −− 186 A −− 187a S − 198 V − 204 A −− 211 V − 215 P −− 251e Q −260 A − 265 A − 339 V − 365 A −− 383k E − 404 G −− 417 R −− Conphys — 0

TABLE 8 Specific Amended in position Substitution Tm (° C.) activity atpH no. into (DSC) 5.0 (U/mg) 43 and 51 and 220 and 51A, 220N, 84.7 105244 and 264 and 302 244D, 264I, and 337 and 352 and 302H, 337T, 373352K, 373A, 43T as above plus 80 as above plus 85.7 180 80A Conphys —78.1 30

Example 7

Cloning of a Phytase of Cladorrhinum foecundissimum

DNA encoding a phytase from Cladorrhinum foecundissimum CBS 427.97 hasbeen cloned, and the enzyme isolated and purified, essentially asdescribed in WO 98/28409.

FIG. 2 shows the DNA sequence of the HindIII/XbaI cloned PCR product inpA2phy8. The cloned PCR product is amplified from the genomic regionencoding Cladorrhinum foecundissimum CBS 427.97 phyA gene. The putativeintron is indicated by double underline of the excision-ligation pointsin accordance with the GT-AG rule (R. Breathnach et al., 1978, Proc.Natl. Acad. Sci. USA 75, pp. 4853-4857). The restrictions sites used forcloning are underlined.

According to the SignalP V1.1 prediction (Henrik Nielsen, JacobEngelbrecht, Stren Brunak and Gunnar von Heijne: “Identification ofprokaryotic and eukaryotic signal peptides and prediction of theircleavage sites,” Protein Engineering 10, 1-6 (1997)), the signal peptidepart of the enzyme corresponds to amino acids nos. 1-15, accordingly themature enzyme is amino acids nos. 16-495.

The enzyme exhibits a pH optimum around pH 6 with no activity at the lowpH (pH 3), but significant activity up until pH 7.5; thus it is a morealkaline phytase as compared to the Aspergillus ficuum phytase.

A temperature optimum around 60° C. was found at pH 5.5. Thus, thisphytase is more thermostable than the A. ficuum phytase.

Example 8 Alignment of a New Model Phytase According to FIG. 1

The phytase sequence of Cladorrhinum foecundissimum as disclosed inExample 7 is compared with the 13 model phytases of FIG. 1 using GAPversion 8 referred to above with a GAP weight of 3.000 and a GAPlengthweight of 0.100. Complete amino acid sequences are compared. The M_(—) thermophila phytase sequence turns up to be the most homologoussequence, showing a degree of similarity to the C. foecundissimumsequence of 70.86%.

Still using the GAP program and the parameters mentioned above, thephytase sequence “C _(—) foecundissimum” is now aligned to the“M-thermophila” phytase—see FIG. 3. The average match is 0.540; theaverage mismatch −0.396; quality 445.2; length 505; ratio 0.914; gaps 9;percent similarity 70.860; percent identity 53.878.

In a next step, see FIGS. 4A-4D, the C _(—) foecundissimum is pasted (orit could simply be written) onto the alignment of FIG. 1 as the bottomrow, ensuring that those amino acid residues which according to thealignment at FIG. 3 are identical (indicated by a vertical line) orsimilar (indicated by one or two dots) are placed above each other. At 5places along the sequence, the C _(—) foecundissimum sequence comprises“excess” amino acid residues, which the alignment of FIG. 1 does notmake room for. At FIGS. 4A-4D, these excess residues are transferredonto a next row (but they can be included in the multiple alignment andnumbered as described previously in the position numbering relatedparagraphs (using the denotations a, b, c etc.).

Corresponding variants of the phytase of C _(—) foecundissimum are theneasily deduced on the basis of FIGS. 4A-4D. Some examples: The variantsgenerally designated “80K,A” and “43T” in C _(—) foecundissimumcorrespond to “K80A” and “Q43T,” respectively.

1-85. (canceled)
 86. A modified phytase comprising a mutation in anamino acid sequence of a phytase, wherein the modified phytase hasphytase activity and the mutation is at one or more positions selectedfrom the group consisting of: 24; 27; 31; 33; 39; 40; 41; 42; 46; 49;56; 59; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 81; 82; 84; 116;117; 119; 120; 121; 122; 123; 124; 125; 127; 128; 132; 149; 150; 151;152; 155; 156; 157; 158; 159; 160; 161; 162; 163; 170f; 170g; 171; 184;185; 187; 190; 191; 192; 193; 194; 200; 201; 201a; 201b; 201c; 201d;201f; 202; 223; 228; 232; 233; 235; 236; 237; 239; 243; 246; 253; 256;271; 272; 274; 275; 276; 277; 279; 280; 283; 285; 287; 288; 292, 293;304; 332; 333; 334; 335; 336; 338; 341; 342; 343; 348; 349; 362; 364;367; 368; 369; 370; 371; 372; 374; 375; 376; 387; 393; 394; 396; 409;412; 413; 421; and 431, wherein each position corresponds to theposition of the amino acid sequence of the mature P. lycii phytase (SEQID NO: 7).
 87. The modified phytase of claim 86, wherein the mutation isselected from the group consisting of: 24C; 27P; 31Y; 33C; 39H,S,Q;40L,N; 42S,G; 49P; 56P; 58D,K,A; 59G; 69Q; 75W,F; 78D,S; 81A,G,Q,E; 82T;84I,Y,Q,V; 116S; 119E; 120L; 122A; 123N,Q,T; 125M,S; 127Q,E,N; 128A,S,T;132F,I,L; 151A,S; 152G; 157V; 158D,A; 159T; 160A,S; 161T,N; 162N; 163W;170fH; 170gA; 171N; 184Q,S,P; 185S; 187A; 190A,P; 193S; 194S,T; 200G,V;201D,E; 201a( ); 201b( ); 201c( ); 201d( ); 201f( ); 202S,A; 223H,D;228N; 232T; 233E; 235Y,L,T; 236Y,N; 237F; 246V; 253P; 256D; 271D,N;275F,Y; 280A,P; 283P; 287A,T; 288L,I,F; 292F,Y; 293A,V; 304P,A; 332F;336S; 338I; 343A,S,F,I,L; 348Y; 349P; 362P; 364W,F; 367A,K; 368K;369I,L; 370V; 374S,A; 375H; 376M; 387P; 393V; 396R; 409R; 412R; 421F,Y;and 431E.
 88. The modified phytase of claim 86, wherein the phytase isan ascomycete phytase.
 89. The modified phytase of claim 88, wherein thephytase is an Aspergillus phytase.
 90. The modified phytase of claim 89,wherein the phytase is an Aspergillus ficuum, Aspergillus fumigatus,Aspergillus nidulans, Aspergillus niger, or Aspergillus terreus phytase.91. The modified phytase of claim 90, wherein the phytase is anAspergillus terreus, CBS 116.46 phytase.
 92. The modified phytase ofclaim 86, wherein the phytase is a Myceliophthora thermophila,Talaromyces thermophilus, or Thermomyces lanuginosus phytase.
 93. Themodified phytase of claim 92, wherein the phytase is a Myceliophthorathermophila, ATCC 34625 or ATCC 74340 phytase.
 94. The modified phytaseof claim 88, wherein the phytase is a Talaromyces thermophilus, ATCC20186 or ATCC 74338 phytase.
 95. The modified phytase of claim 88,wherein the phytase is a Thermomyces lanuginosus, NRRL B21527 phytase.96. The modified phytase of claim 86, wherein the phytase is anascomycete consensus phytase sequence.
 97. The modified phytase of claim86, wherein the phytase is a basidiomycete phytase.
 98. The modifiedphytase of claim 97, wherein the phytase is an Agrocybe pediades,Paxillus involutus, Peniophora lycii, or Trametes pubescens phytase. 99.The modified phytase of claim 98, wherein the phytase is a Paxillusinvolutus, CBS 100231 phytase.
 100. The modified phytase of claim 99,wherein the phytase is a Paxillus involutus, CBS 100231 Phy-A2 phytase.101. The modified phytase of claim 98, wherein the phytase is a Trametespubescens, CBS 100232 phytase.
 102. A feed or food comprising a modifiedphytase of claim
 86. 103. A composition comprising a modified phytase ofclaim
 86. 104. A process for reducing phytate levels in animal manurecomprising feeding an animal with an effective amount of the feed ofclaim 102.